Our goal is to understand how Golgi stacks are generated. An attractive model is that new Golgi elements arise from the COPII transport vesicles that are produced at transitional ER (tER) sites. Thus, characterizing the tER will be crucial for understanding the Golgi. For example, Golgi distribution in budding yeasts seems to be a consequence of tER distribution. In Saccharomyces cerevisiae, Golgi cisternae are dispersed throughout the cytoplasm and the entire ER functions as tER; whereas in Pichia pastoris, coherent Golgi stacks are located next to discrete tER sites. These two yeasts will be analyzed through a combination of genetics, biochemistry and microscopy to elucidate the mechanisms that define the tER-Golgi system. Our Specific Aims are: (1) To characterize S. cerevisiae mutants defective in Golgi inheritance. This study will test the idea that Golgi inheritance is a byproduct of ER inheritance. (2) To isolate P. pastoris mutants with abnormal tER organization. Such mutants will lead us to the proteins that define tER sites, and will reveal whether Golgi distribution is indeed a consequence of tER distribution. (3) To determine how P. pastoris Sec12p is localized to tER sites. Because Sec12p is thought to be the "master regulator" of the COPII assembly pathway, analyzing the localization mechanism of this protein will yield insights into tER formation. Abnormal Golgi function is a causative agent in many diseases, including cancer as well as various inherited and autoimmune diseases. Treatment of these diseases will require a cell biological understanding of the processes that establish and maintain Golgi organization. Our work aims to elucidate these basic principles of Golgi biogenesis.